Butyric acid is used in butter and rum flavors, and its ethanol ester (ethyl butyrate) is used in fruit flavors. Other esters of butyric acid are also used in flavors. The production of such natural flavor ingredients by fermentation has become a desirable alternative to their physical extraction from plants because it avoids the disadvantages of such extraction while retaining the recognized "natural" designation, based on the ruling by the FDA (21 CFR 101.22.a.3) that products of fermentation are considered natural provided that the starting substrate is a natural material such as glucose or sucrose.
The production of butyric acid by fermentation is well known. Often acetic acid is also produced in these fermentations; acetic acid and several of its esters (particularly ethyl acetate) are also of interest to the flavor industry. Usually a member of the genus Clostridium is used for the butyric acid fermentation, and the fermentation is conducted anaerobically. Substrates comprising sugars such as glucose, sucrose, and fructose, or carbohydrates that break down into such sugars are conventionally used, for example, in batch, semi-batch ("fed-batch") or continuous operations.
Fermentation processes that can produce butyric acid are described, for instance, in U.S. Pat. Nos. 4,539,293 and 4,814,273, and in an article published in Appl. Microbiol. Biotechnol. (34, 2, 172-77) 1990, by D. Michel Savin, D. R. Marchal, and J. P. Vandecasteele of the Division Biotechnoloqie et Environnement, Institut Francais du Petrole, that describes the metabolic behavior and production performance of Clostridium tyrobutyricum in a continuous culture.
U.S. Pat. No. 4,814,273 describes the fermentation in batch mode of a lactate salt to butyrate salt using Butyribacterium methylotrophicum (B. methylotrophicum), and following that fermentation with a second fermentation in the same mixture that converts a carbohydrate to lactic acid using a Lactobaccillus, preferably after removing the cells of the microorganism used in the first step. Productivity is usually higher with continuous operation (as suggested by the good results reported by Michel Savin et al. in the above literature publication), because unproductive downtime for repeatedly draining the fermentor, refilling the fermentor, and growing the culture is not required.
For continuous operation, the conventional arrangement is a chemostat, in which nutrient medium is fed to a stirred fermentor tank at a controlled rate and broth is removed at the same rate, so that the level in the fermentor remains constant. The dilution rate (expressed as liters.liter.sup.-1.hr.sup.-1, or simply hr.sup.-1) must not exceed the maximum growth rate of the organism, and the formation of the desired product must be at least partially associated with the growth of the microorganism. Complex nutrient supplements (e.g., yeast extract) are conventionally used, as additives or in complex media compositions, to obtain a high yield of product, as in U.S. Pat. No. 4,814,273, although they may have some disadvantages; for example, these complex materials, which have variable compositions, can support growth of a wide variety of organisms, so that they render the fermentor more susceptible to unpredictable contamination. Furthermore, they usually are not completely soluble, which can lead to abrasion problems with heat exchangers, pumps, and fermentor agitator blades, and makes the medium unsuitable for sterilization by filtration, which is sometimes desirable to reduce energy costs or if the medium contains heat-labile components.
For these reasons a nutrient substrate essentially comprising a compound having a chemically definable structure, such as glucose or sucrose, and all other medium components having a chemically definable structure may be desirable, although the yield of the desired product may not be as high and small amounts of specific additional nutrients may have to be added.
The published disclosures of continuous fermentation work do not address the problems that are encountered in the conventional procedures used to start up a continuous butyric acid fermentation process. The fermentor is conventionally operated in batch mode until the concentration of the microorganism is sufficiently high, then addition of the fermentation medium (feedstock) and removal of the medium containing the butyric acid product (fermentation broth) at a continuous rate that maintains a constant volume of medium in the fermentor is continued. However, when feeding of the medium begins, it causes drastic changes in the established balance of the ingredients of the fermentation broth; the butyric and acetic acid concentrations typically decrease and the residual substrate concentration correspondingly increases. As much as several days may be required before the proper concentrations of the products in the medium are re-established. In a commercial process this transitory reduction in butyric acid yield can be very costly, and the presence of excessive substrate can interfere with downstream purification of the products.
There is clearly a need for a method of making a smooth transition from batch to continuous operating mode, without such undesirable changes in the established balance of the ingredients.